Selection of a novel and highly specific tumor necrosis factor alpha (TNFalpha) antagonist: insight from the crystal structure of the antagonist-TNFalpha complex

Abstract

Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.

FIGURE 1.

Schematic overview of the selection and maturation process. The red X indicates the amino acid position that was randomized in a given library. The terms Primary lib and mat lib refer to primary and maturation libraries, respectively. The sequences of CTLD libraries used as a basis for the next round of selection are shown in bold. a, amino acids maintained from the primary clone to TN-2-B-1-C31. b, amino acids maintained from the first round maturation clone to TN-2-B-1-C31. c, amino acids maintained from the second round maturation clone to TN-2-B1-C31.

FIGURE 2.

A, overview of hTNFα-CTLD complex structure. The hTNFα secondary structure is shown as green ribbon, combined with a transparent surface representation. The part of the hTNFα surface involved in interactions with CTLD is shown in space-fill coloring according to electrostatic potential. The antagonist is depicted as cyan ribbon, except for the randomized part, which is shown in magenta. B and C, close up of the hTNFα-CTLD interactions. Amino acid labels in red refer to CTLD, and amino acid labels in black refer to hTNFα. D, reconstruction of trimeric version of the TN2-B1-C31 using the structure of natural trimeric tetranectin as a model. This was done by substituting the structure of the CTLD domain found in the structure of natural tetranectin (PDB ID 1HTN) with the structure of the TN2-B1-C31 clone in complex with hTNFα. Next we added the symmetry mates of hTNFα to reconstruct the naturally occurring trimeric hTNFα (panel E).